34th Annual Student Research Conference
The Effect of Two Subsequent Purifications on DBF Using Immobilized Metal Affinity Column Chromatography
Derek M. Empson
Dr. Cassidy Dobson, Faculty Mentor
Disulfide Bond Forming Enzyme (DBF) is a chaperone protein able to refold incorrectly paired disulfide bonds. DBF is unique because it lacks any cysteine residues in its structure. This lack of cysteines indicates a novel mechanism of action compared to other chaperones responsible for disulfide bond rearrangement. In order to investigate the mechanism of DBF, it is recombinantly over-expressed in E. coli in a pET-28 vector. However this expression also contains other proteins that are endogenous to E. coli, meaning purification is needed to get only DBF. Immobilized metal affinity column chromatography (IMAC) is the chosen method of purification because of the Histidine tag on the pET-28 vector. The extent of purification is then analyzed by SDS-PAGE. The purpose of this investigation is to determine if two subsequent purifications would be significantly more effective than only one in purifying DBF.
Keywords: DBF, IMAC, Proteins, E. Coli, SDS-PAGE
Topic(s):Biochemistry and Molecular Biology
Presentation Type: Asynchronous Virtual Oral Presentation
Session: 3-4
Location: https://flipgrid.com/f86d186b
Time: 0:00